The absorptive and secretory capacity of epithelial cells of the epididymis and vas deferens (VD) creates the appropriate environment for spermatozoa as they mature and are stored. The lumen of these organs is maintained acidic compared to blood and has a low bicarbonate concentration. These factors are involved in sperm maturation and the maintenance of sperm in a quiescent state during their storage period. We propose that Na/H exchanger-rich (principal) cells actively absorb bicarbonate in the proximal regions of the epididymis and that H+ATPase-rich (clear) cells are responsible for net proton secretion in the distal epididymis and vas deferens. Specific aim 1 will characterize the acid/base transporters involved in luminal acidification in the initial segments of the epididymis, and in the cauda epididymis and VD (H+ATPase, Na+/H+ exchanger, NBC1, NBC3, AE2). Specific aim 2 will examine the molecular mechanisms responsible for the regulation of proton secretion and bicarbonate reabsorption. Specifically, we will study the role of SNARE proteins, the actin-severing protein, gelsolin, and the newly identified "bicarbonate sensor" adenynyl cyclase, sAC, in the recycling of H+ATPase-containing vesicles. The role of the Na/H exchange regulatory factor, NHE-RF, and sAC will also be examined in bicarbonate reabsorption, possibly via CAMP-modulation of the Na/H exchanger. Specific aim 3 will examine the hormonal regulation of luminal acidification in the epididymis and VD. Short-term and long-term regulation by steroid hormones (aldosterone, testosterone, estrogens) and by angiotensin II will be studied. These studies will be carried out using a multidisciplinary approach (proton-selective self-referencing extracellular electrode, intracellular pH measurements, light microscopy and EM immunocytochemistry, western blotting, in situ hybridization, laser capture microdissection and real time RT-PCR), and will help to unravel the mechanisms underlying acid/base transport in the organs where spermatozoa mature and are stored. The ultimate aim of this application is to better understand the physiology and pathophysiology of male fertility, because a defect in the acidification capacity of the epididymis and vas deferens might result in deficient sperm maturation and motility, leading to lower fertility. Project 12 will benefit from constant interaction with the other projects of this Program, especially Projects 7 and 9, which propose to characterize AQP2 recycling, and the mechanisms for acidification-dependent endosomal regulation, respectively.